Polymer for binding amine containing ligands and uses thereof

ABSTRACT

Reaction of a dialdehyde, particularly phthaldialdehyde (I), with R-Z where Z is a nucleophilic group (preferably SH) and R is polymerisable (e.g. allyl) gives a reactive thioacetal (V) which can react with an amine ligand L-NH2 to produce an isoindole (IV) which may be fluorescent. At some stage, generally before interaction with L-NH2, the R groups are polymerised, possibly leading to self-assembly of the polymer on a metal or SH-bearing surface. Such a coated surface is useful as a transducer in assays or as a binding medium e.g. for chromatography.

TECHNICAL FIELD

The present invention relates to a polymer capable of binding amineligands, to its preparation, and to uses including assays andchromatography.

There is an ever-increasing need for the measurement and quantificationof mankind's activities. Sensors are partially fulfilling thisrequirement though sensors for many applications are not readilyavailable in a form suitable for commercialisation.

One particular class of sensors, that is bio- and chemo-recognitionsensors, requires the integration of ligands (recognition elements) to atransducer. The integration should normally enable changes in therecognition element caused by interaction with a corresponding analyteto be converted into a measurable signal via the transducer.

The immobilisation of the ligands to the surface of a suitabletransducer is a necessary step in sensor manufacturing. Desirableproperties of this process include: placement of appropriate amounts ofligands on detector surface using simple procedures, maintenance ofbiorecognition activity of the ligands after immobilisation, andminimisation of non-specific interactions between the sample and therecognition element or other parts of the sensor.

BACKGROUND ART

Recently a wide variety of methods have been discovered for attachingdifferent ligands to transducers in order to create effective andsensitive bio- and chemosensors.

Examples are disclosed in U.S. Pat. No. 4177038, U.S. Pat. No. 4250267,U.S. Pat. No. 4784962, U.S. Pat. No. 5242828, U.S. Pat. No. 5436161 andLöfås, S., Johnsson, B., Tegendal, K., Rönnberg, I., “Dextran modifiedgold surfaces for surface plasmon resonance sensors: immunoreactivity ofimmobilised antibodies and antibody-surface interaction studies”,(1993), Colloids and Surfaces B: Biointerfaces, 1: 83-89.

Also of interest is Simons, S., Jr. et al. “Reaction of O-Phthalaldehydeand Thiols With Primary Amines; Formation of 1-Alkyl (and Aryl)Thio-.alpha.-Akylisoindoles”, (1978), J. Org. Chem., 43 (14): 2886-2896.

A common approach is the use of transducer surfaces modified withhydrogel layers (U.S. Pat. No. 5436161), such as carboxymethyl-dextran.Typically, hydrogel polymers are covalently immobilized to a transducersurface to form a thin hydrogel layer. Ligands can be covalentlyimmobilised to the hydrogel polymer using a chemical activation. Suchmulti-step process places a significant overhead cost on anymass-manufacture of a sensor device. An alternative approach, which caneliminate this disadvantage, would be of considerable benefit.

DISCLOSURE OF INVENTION

In a first aspect the invention provides a method of immobilising anamino-group containing ligand L—NH₂ by means of a dialdehyde componentOHC—X—CHO and a polymerisable component R—Z wherein R is a polymerisablemoiety and Z is selected from —SH, —S-alkyl, —CN and —SO₂ by carryingout the following reactions simultaneously and/or sequentially in anychemically feasible order:

i) polymerisation of the polymerisable moieties R, optionally togetherwith one or more comonomers;

ii) reaction of a component containing —Z with the dialdehyde component;

iii) reaction of L—NH₂ with the dialdehyde component or with the productof reaction (ii).

The dialdehyde component is preferably a 1,4-dialdehyde, generallyconjugated

generally forming part of an aromatic ring system, e.g. it may beo-phthaldialdehyde (I):

Conjugation may be desirable to give a product detectable by opticalmethods, e.g. involving fluorescence.

The polymerisable moiety R generally contains one or more carbon-carbonmultiple bonds. For example, R—Z may be allyl mercaptan.

Polymerisation may involve comonomers as well as the R moieties. Theycan be used to ‘dilute’ the Z groups in the polymer. Particularly when Zis —SH, such dilution may be desirable to reduce the occurrence ofcross-linking through Z—Z (e.g. disulphide) bridges. Comonomers can alsobe used to regulate polymer solubility, to suit particular applications.

Particularly when Z is —SH or —S-alkyl, the reaction may be suitable forachieving self-assembly of a polymer on a suitable surface (particularlya metal surface (especially noble metal) or a surface having —SHgroups). Thus a preferred class of embodiment is based on the ability ofa synthetic polymer, containing thioacetal groups formed by a mercaptogroup and phthalic dialdehyde to self-assemble on a metal surface andbind amino containing ligands to form a fluorescent complex. The polymercan be synthesised using ion, radical polymerisation orpolycondensation. It is preferable that at least one of the monomersused for polymerisation contains free SH groups. In another variantpolymer can be first produced using an SH-group containing monomer andsubsequently treated with dialdehyde to form a thioacetal. IL is alsopossible to use CN and SO₂-containing monomers instead or simultaneouslywith SH-containing monomer for the polymer preparation.

Surfaces coated with a polymer of the invention may be microtitreplates, wells, transducers (e.g. for surface plasmon resonance orelectrochemical devices), or binding materials e.g. for chromatography.

A polymer of the invention may comprise units of formula II:

where R′ is derived from polymerisation of an R group. The unit

is preferably provided by an aromatic ring system, e.g. being

optionally substituted and/or fused to form a polycycle. A polymer ofthe invention incorporating L—NH₂ may comprise units of formula III:

where

corresponds to

in formula II, e.g. being

(optionally substituted and/or fused.

The polymers described above can be used in affinity chromatography orsensors. Such a polymer containing SH groups may adsorb on a metal orSH-containing surface (e.g. during the synthesis) forming a homogeneous,stable coating. It is possible to make first a surface coating with apolymer of RSH, with following treatment of this polymer usingdialdehyde, preferably phthalic dialdehyde and next amino-containingligand. Again, ligand can be immobilised by simultaneous addition of thedesired compound with dialdehyde to the SH-containing polymer. Species,such as cells, enzymes, viruses, fungi, antibodies, proteins, peptides,amino acids, nucleic acids and derivatives or mixtures can be easilyimmobilised on the polymer, synthesised as described above. Theimmobilised species may itself serve for binding a second type ofligand. Binding the second type of ligand may affect measurableproperties, e.g. fluorescence, so that the binding may be detected. Theligands of first and second type may constitute specific binding pairs,e.g. antibody-antigen.

Ligand immobilisation on the polymer surface includes one-stepinteraction between thioacetal and amino group without chemicalactivation of the polymer or ligand functional groups. Formation offluorescent complexes can be used to monitor binding. The isoindolecomplex formed by thioacetal with ligand amino groups was found to bevery stable, which permits the use of strong acidic conditions forsurface regeneration.

The homologous aromatic dialdehyde, o-phthaldialdehyde, or thioacetal isessentially nonfluorescent until reacted with primary amine in thepresence of excess cyanide or mercaptan to yield a fluorescentisoindole. Monitoring of the polymer fluorescence provides anopportunity to directly control the amount of the bound substances tothe polymer surface, which can be further used in sensors and assays.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows possible reaction schemes usable in the invention;

FIG. 2 depicts a dependance of the OPA-AM-polymer emission spectra fordifferent ammonium hydroxide concentrations (1-100 mM). Dotted line-control without ammonium hydroxide. Reaction mixture is 3 mg/ml polymerin 1:1 mixture of acetonitrile and 0.1 M phosphate buffer, pH 7.8.Incubation time is 30 min.

FIG. 3 depicts a dependence of OPA-AM-polymer response for 100 mMammonia on pH.

FIG. 4 depicts a change in the emission spectra intensity of theOPA-AM-polymer (1(lamda)_(exc) 355 nm) as response to the addition ofhorseradish peroxidase.

FIG. 5 depicts a polymer deposition on gold surface using flow system.

FIG. 6 depicts a covalent binding of human immunoglobulin to theOPA-AM-polymer coated surface followed by anti-human immunoglobulinbinding.

FIG. 7 depicts a regeneration of surface coated with polymer with 0.1%SDS/10 mM HCl.

FIG. 8 depicts a displacement of anti- human immunoglogulin (HIGg) bythe human immunoglobulin from the prism surface coated by OPA-AM-polymerwith following regeneration. Concentrations of HIGg and anti-HIGg-10mg/ml.

FIG. 9 is a bar chart showing the binding capacity of the OPA-AM polymerfor different proteins.

MODES FOR CARRYING OUT THE INVENTION

FIG. 1 shows some examples of reaction types that may be used in theinvention.

Reaction (i) shows the interaction of o-phthaldialdehyde (“OPA”), (I)with a polymerisable thiol R—SH and an amine ligand L—NH₂ to produce afluorescent isoindole (IV). Reactions (ii) and (iii) show this processcarried out in sequential stages: reaction of OPA (I) with the thiol toform a thioacetal (V), which subsequently reacts with the amine ligand.

The preparation of a polymer embodying the invention and someexperiments carried out with it will now be described.

1. Preparation of the Polymer

134 mg of OPA and 82 mg of allyl mercaptan (AM) were dissolved in 2 mlof (2-hydroxyethyl)methacrylate and 2 ml of acetonitrile. 50 mg AIBINwas used as a catalyst for this reaction. Reaction mixture was incubatedovernight at 80°. Polymer (OPA-AM) was dissolved in ethanol and 0.1 Msodium phosphate buffer pH 8.0 was added. The resulting pellet wascollected and washed with water using glass filter.

2. Fluorometry With Reactive Polymer

30 mg of OPA-AM polymer was dissolved in 10 ml of acetonitrile/H₂O(1:1). To adjust mixture for basic pH 30 ml 4% NaOH was added.Fluorescence measurements were performed with RF-5301 PCSpectrofluorophotometer (Shimadzu, Japan).

The polymer was excited at wavelength 355 nm and emission spectrum wasmeasured. It was found that fluorescence intensity depends on thepresence of amino-groups in the sample (see FIG. 2), incubation time(not shown) and pH (see FIG. 3).

The same experiments were carried out using horseradish peroxidasegiving similar results (FIG. 4.).

3. Application of OPA-AM Polymer for Ligand Immobilisation in SurfacePlasmon Resonance

Glass prisms coated with >>50 nm gold layer were immersed in the polymersolution in methanol (1 mg/ml) for 5 min, washed twice with methanol andwater.

In another variation of the method the polymer solution in methanol wasrun through the flow cell (20 ml volume) with flow rate 5 ml/min for 10min. Methanol was then run for 10 min to remove excess polymer. Theshift in position of reflective curve minimum (A_(SPR)) is a result ofpolymer immobilisation (FIG. 5).

100 ml human IgG solution (0.1 mg/ml, 100 mM sodium borate buffer, pH9.0) was injected and rate of immobilisation was measured using anglescanning. The displacement of the reflective curve minimum position wasmeasured.

The adsorption of the second antibodies specific for covalentlyimmobilised HIGg (anti-HIGg) was observed (FIG. 6). The surface wasregenerated with mixture of 0.1% SDS/10 mM HCl (FIG. 7).

The time scanning was applied to measure binding/displacement events(FIG. 8). It was found that OPA-AM matrix creates reactive surface,which is suitable for optic transducers. It is stable upon storage,simple to prepare and is suitable for mass-production of biosensordevices.

Protein immobilization. The binding capacity of the polymer wasdemonstrated with several proteins: microperoxidase (FW=1 kD),cytochrome C (FW=12.4 kD), horseradish peroxidase (HRP) (FW=44 kD),bovine serum albumin (BSA) (FW=66 kD) and haemoglobin (FW=67 kD). 10 mgof polymer was incubated with 400 ml of protein solution (5 mg/ml) in 10mM HEPES buffer, pH 8.6 for 18 hours. The protein concentration beforeand after sorption was measured spectrophotometrically using a BCAmethod [cf Osnes T, Sandstad O, Skar V, Osnes M, Kierulf P, Scandinavianjournal of clinical & laboratory investigation 1993; 53 (7): 757-763.]and calculated using calibration curves obtained individually for eachprotein. It was calculated that 1 g of the polymer can bind 0.6 mg ofthe BSA, 0.55 mg cytochrome C, 0.2 mg microperoxidase, 0,5 mghorseradish peroxidase and 1 mg haemoglobin (Figure). The bindingcapacity of the polymer and sensitivity of the fluorescent detectiontherefore depend on the number of amines available—a function of boththe protein's structure and its amino acid composition. Theimmobilisation rate was found comparable with commercial sorbents usedfor protein immobilisation like activated CN Sepharose 4B (Pharmacia,Sweden). It is anticipated that the polymer can be used as effectivealternative immobilisation matrix in affinity chromatography forimmobilisation of low-weight organic amines, proteins and nucleic acidsas well as sensor/assay components for primary amines detection.

What is claimed is:
 1. A polymer comprising units of the formula

wherein R′ is derived form a polymerizable moiety containing one or morecarbon-carbon multiple bonds.
 2. The polymer according to claim 1wherein

is an aromatic ring system.
 3. A binding medium comprising a carriermaterial having a surface which bears a polymer according to claim
 1. 4.The binding medium according to claim 3 which is a stationary phase forchromatography.
 5. A chromatography column or plate having a stationaryphase according to claim
 4. 6. A biosensor device having a surfacecovered with the binding medium as claimed in claim
 3. 7. A method ofassaying a sample for ligands, comprising: exposing the surface of thedevice of claim 3 bearing said binding medium to a sample suspected ofcontaining said ligands; and monitoring optical properties of saidbinding medium.
 8. The method according to claim 7, wherein the carriermaterial bears a polymer comprising units of the formula

where R′ is derived from a polymerizable moiety containing one or morecarbon-carbon multiple bonds and said polymer serves for bindingamino-group containing ligands.
 9. The binding medium according to claim3, wherein the carrier material bears a polymer comprising units of theformula

where R′ is derived from a polymerizable moiety containing one or morecarbon-carbon multiple bonds and said polymer serves for bindingamino-group containing ligands.
 10. A polymer comprising units of theformula

wherein R′ is derived form a polymerizable moiety containing one or morecarbon-carbon multiple bonds and

is derived from a ligand L—NH₂ which is selected from the groupconsisting of an amino acid, peptide, protein, nucleic acid andderivatives thereof.
 11. A polymer according to claim 10 wherein

is an aromatic ring system.
 12. A binding medium comprising a carriermaterial having a surface which bears a polymer according to claim 10.13. A binding medium according to claim 12, which is a stationary phasefor chromatography.
 14. A chromatography column or plate having astationary phase according to claim
 13. 15. A method of immobilizing anamino-group containing ligand L—NH₂, comprising: polymerizing a monomerR—Z, wherein R is a polymerizable moiety and Z is a functional groupselected from the group consisting of —SH, —S-alkyl, —CN and —SO₂,optionally in the presence of a comonomer; reacting a dialdehydecomponent of the formula OHC—X—CHO, wherein X is a linking group, withsaid functional group; and then reacting a ligand L—NH₂ with thedialdehyde modified polymer.
 16. The method of claim 15, wherein thedialdehyde component is a 1,4-dialdehyde.
 17. The method of claim 15,wherein the dialdehyde component is of the form:

wherein the partial structure A—C═C—B defines an aromatic ring system.18. The method of claim 17, wherein the dialdehyde component iso-phthaldialdehyde.
 19. The method according to claim 15, wherein Rcontains one or more carbon-carbon double or triple bonds.
 20. Themethod according to claim 15, wherein said polymerizable component is athiol R—SH.
 21. A method of immobilizing an amino-group containingligand L—NH₂, comprising: reacting a ligand L—NH₂ and a dialdehydecomponent of the formula OHC—X—CHO, wherein X is a linking group, in thepresence of a polymerizable monomer R—Z, wherein R is a polymerizablemoiety and Z is a functional group selected from the group consistingof—SH, —S-alkyl, —CN and —SO₂, optionally in the presence of acomonomer, thereby effectively polymerizing the monomer and optionalcomonomer and simultaneously reacting the ligand L—NH₂ with thedialdehyde to form a product which reacts with the functional group Z ofthe polymerizing monomer R—Z.
 22. The method of claim 21, wherein thedialdehyde component is a 1,4-dialdehyde.
 23. The method of claim 21,wherein the dialdehyde component is of the form:

wherein the partial structure A—C═C—B defines an aromatic ring system.24. The method of claim 23, wherein the dialdehyde component iso-phthaldialdehyde.
 25. The method according to claim 21, wherein Rcontains one or more carbon-carbon double or triple bonds.
 26. Themethod according to claim 21, wherein said polymerizable component is athiol R—SH.
 27. A method of immobilizing an amino-group containingligand L—NH₂, comprising: reacting a ligand L—NH₂ and a dialdehydecomponent of the formula OHC—X—CHO, wherein X is a linking group,thereby forming a reaction product; and then reacting the reactionproduct, which reacts with the functional group Z of polymerizablemonomer R—Z, with (1) a polymer prepared by polymerizing a monomer R—Zand optionally with a copolymerizable comonomer or with (2) a monomerR—Z and optionally with a copolymerizable comonomer, followed bypolymerization of the monomer material, wherein R is a polymerizablemoiety and Z is a functional group selected from the group consisting of—SH, —S-alkyl, —CN and —SO₂.
 28. The method of claim 27, wherein thedialdehyde component is a 1,4-dialdehyde.
 29. The method of claim 27,wherein the dialdehyde component is of the form:

wherein the partial structure A—C=C—B defines an aromatic ring system.30. The method of claim 27, wherein the dialdehyde component iso-phthaldialdehyde.
 31. The method according to claim 27, wherein Rcontains one or more carbon-carbon double or triple bonds.
 32. Themethod according to claim 27, wherein said polymerizable component is athiol R—SH.
 33. A method of immobilizing a ligand on a surface,comprising: adsorbing a polymer prepared by polymerizing a monomer ofthe formula R—SH, optionally in the presence of a comonomer, on a metalsurface; reacting a dialdehyde of the formula OHC—X—CHO, wherein X is alinking group, with the surface adsorbed polymer; and then reacting aligand L—NH₂ with the reacted polymer, thereby immobilizing the ligandon the surface.
 34. The method according to claim 33, wherein theimmobilized ligand fluoresces which is detected by a fluorescent lightdetecting device.
 35. A method of immobilizing a ligand on a surface,comprising: adsorbing a monomer of the formula R—SH, optionally in thepresence of a comonomer, on a metal surface with the presence of adialdehyde of the formula OHC—X—CHO, wherein X is a linking group, andthen effecting polymerization of the (co)monomer and reaction of thedialdehyde with the —SH functional groups of the polymer; and thenreacting a ligand L—NH₂ with the reacted polymer, thereby immobilizingthe ligand on the surface.
 36. The method according to claim 35, whereinthe immobilized ligand fluoresces which is detected by a fluorescentlight detecting device.